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					IOSR Journal of Pharmacy and Biological Sciences (IOSRJPBS)
ISSN : 2278-3008 Volume 1, Issue (July-August 2012), PP 09-16
www.iosrjournals.org

      New Generation Expression Host System- Aiming high for
          commercial production of recombinant protein
                         1
                             Mahendra K. Verma, 2Yogendra K. Verma
                     1
                      Department of Biotechnology, R.V.R. & J.C. College of Engineering
                                  Guntur, Andhra Pradesh, India- 522 019
                  2
                   Department of aerobic Microbiology, All India Institute of Medical Science
                                          New Delhi, India-110029

Abstract- The global requirements of recombinant protein have increased exponentially in last one decade in
various aspects. The commercial production of recombinant proteins requires an ideal host expression system
which effectively meets the demand. Numerous host expression systems have been used to achieve level of
expression. Escherichia coli BL21 (DE3) have been used ideally for expression of numbers of recombinant
proteins from many decades. Though, Escherichia coli Bl21 is quite enough to express recombinant protein in
higher fold but often get fails to achieve over expression of protein as essential requirement for today demand.
Recently many engineered strains have been implemented in order to bring expression level as per requirement.
The complications which arise in the expression of recombinant protein are mainly due to codon bias or
inability of vector promoter to utilize host polymerase; while for over-expression of recombinant proteins often
inhibit itself due to protein toxicity in conventional expression host systems. Escherichia coli C41 (DE3),
Escherichia coli C43 (DE3) and Escherichia coli Rosetta are the refined and engineered strains which have
shown over-expression of recombinant protein in recent time. These strains are designed to overcome the
complication often limits conventional expression system.

Keywords: Codon bias, promoter, Expression, host expression system, Recombinant protein

                                            1. Introduction
          The advancement in technologies in biological science, molecular biology, recombinant DNA
technology and protein engineering have equipped human to understand biomolecules at molecular level and
refine them as per requirements[1]. These refined molecules have been used significantly in medicine, industrial
application and for analytical applications since long time. The protein molecules which essentially used as
enzymes have made many revolutions in medical filed and more ever in industry. Enzymes which are basically
proteins have enormous capability of catalysis, which have been employed for the treatment of various diseases,
optimization of different conventional industrial process and in design of many diagnostic [2, 3]. To achieve
following, enzyme must produced in sufficient quantity with intact activity [4]. To meet the demand in
industrial sector and medical application different methods were opted for these amazing molecules, more
specifically molecular cloning and expression in microbial host systems [5,6]. The benefits of microbial host
systems, they are easy to grow and manipulate as per requirements. Rather than prokaryotic protein, eukaryotic
protein exhibits even more complications while expressions in the prokaryotic host system. This is due to codon
bias which interferes in process of translation while selecting t-RNA form amino acid pool as difference in
codon and lacking of post translational machinery [7, 8]. Recently few host system have been designed to
overcome with these complication like Pichia pastoris. An alternative approach to express the eukaryotic
protein is codon optimization which needs few universal codons especially designed for the protein from
eukaryotic source [9, 10].

                         2. Commercial Demand of recombinant proteins
         The commercial application of recombinant proteins is ubiquitous, protein as enzymes in Industrial
applications; Food Processing, Detergents, Beverages, Leather processing are few sector which have explored
significance of these molecules. The global production of these enzymes is rapidly increasing which needs large
scale production of these potent molecules [11 12 13].




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                         Fig 1: Industrial demand of enzymes worldwide in different sectors

          Simultaneously with industrial applications, recombinant proteins used maximally as therapeutics used
for treatment of number of diseases. These proteins are in clinical use for variety of disease like heart attacks,
strokes, cystic fibrosis, diabetes anemia, and hemophilia etc [14 15].




                        Fig 2: Commercial demand of therapeutic proteins in different forms

                               3. Invitro Protein Expression Strategy
         Protein expression is a natural phenomenon in every living organism to maintain cell viability and
physiology [16, 17]. With the developed technologies the natural process has tuned artificially in specific cells
known as host expression system. The expression of any gene runs under specific nucleotide sequences which
bind RNA polymerase known as promoter sequence [18]. The promoter sequence which provide platform for
RNA polymerase can be classify in natural promoter which usually express protein in threshold level while
certain promoter which basically isolated from viral origin provides high level expression known as strong
promoter [19, 20].

         In case of expression of protein outside the native host one must need a carrier molecule called as
plasmid or vector, possess basic requirement for protein translation. These carrier molecules as per their
application classified cloning vector system used for only cloning and expression vector system which ment for
expression of proteins [21, 22]. The expression vector system essentially contains high strong promoter
generally from viral origin. Frequently used promoters in different expression vector system are CMV and T7
which have been isolated from Cauliflower mosaic virus and T7 bacteriophage respectively [23, 24].




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  Fig 3: Layout of plasmid, three basic requirements for functionality of plasmid system, Origin of replication,
 Promoter allow RNA polymerase to bind and initiate translation and multiple cloning sites for cloning of gene




   Fig 4: A cassette essentially requires for translation of recombinant protein constituted in each of plasmid/
                                             vector used for expression

         The choice of an expression system for the high-level production of recombinant proteins depends on
many factors. These include cell growth characteristics, expression levels, intracellular and extracellular
expression, and biological activity of the target protein [25]. In addition, the selection of a particular expression
system requires a cost down in terms of process, design, and other economic consideration. The combination of
recombinant DNA technology and large-scale culture process has enabled recombinant proteins to be produced
in massive quantities [26]. High cell density culture techniques for culturing E. coli have been developed to
improved productivity and also to provide advantages such as reduced culture volume, enhanced downstream
process, reduced wastewater, lower production costs and reduced investment in equipment [27,28].

                        4. Complications with conventional Expression system
          The complications started while the expression studies for eukaryotic proteins in E. coli expression
systems during 1980 [29, 30]. The E. coli expression system lacks many codons essentially requires for the
expression of eukaryotic protein and also lack post translational modification machinery for biological activity
and some other complex proteins containing multiple disulfide bonds [31, 32, 33]. In addition to codon bias and
lack of post translational modification machinery, many proteins expressed in E. coli got accumulated
intracellular as inactive inclusion bodies [34, 35]. Expressed protein in the form of inclusion bodies further
needs protein renaturation and refolding to retain biological activity, all these transformations are often
complicated and costly while running denaturation and refolding process [36].
          To tackle these complications attributed by E. coli expressions system number of animal and plant
expression system have designed [37, 38]. Though these engineered expression system have made significant
contribution for expression of eukaryotic proteins often failed in E. coli system but again failed to design the
simple and efficient, high-yield, low-cost methods which can be used for the production of proteins in large
amount [39]. Further advancement of technologies especially in protein engineering and recombinant DNA
technology led to development of new generation E. coli expression system competent expression of various
proteins [40]. These engineered E. coli expression system designed with certain molecule; chaperons, foldases
etc allows expressed protein to retain active structure and also helps translocation from cytoplasm to periplasm
and subsequently outside of cell [41, 42].

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                                  5. New Generation Expression system
          In order to meet with industrial demand expression of recombinant protein is best method ever opted.
This method is not only produce protein in high fold also we easy to manipulate [43]. Many expression host
systems were used for production of commercially important enzyme where E. coli BL21 has long history as
ideal host. E. coli BL21 (DE3) strains provide reliable expression of many proteins cloned into T7 expression
vectors (e.g., pET, pRSET etc) [44, 45]. Though, numbers of recombinant protein expressed successfully in BL
21 (DE3) but over expression could not achieve yet [46]. Even in many cases expression is minimal or no
detectable because the recombinant proteins, when expressed, become deleterious or lethal to these standard
BL21 strains. Hence to achieve successful expression and over expression led to design of new engineered
strains capable of over expression beyond these complications [47].
          Recently, many E. coli strains C41 (DE3), C43 (DE3) and E. coli Rosetta strains have been developed
that allow high level of expression to the variety of proteins previously difficult or impossible to express in
bacteria E. coli BL21 [48]. These all strains are genetically engineered ensuring following parameters-

       a-   An ideal expression system for majority of recombinant protein.
       b-   To overcome toxicity exhibited by protein itself.
       c-   Offers expression for both prokaryotic and eukaryotic proteins (Rosetta).
       d-   Easy to regulate and cost factors.

5.1.      Escherichia coli BL Series
          With the advance technologies in molecule biology led to redesign existing BL21 (DE 3) strain in order
to overcome drawback arises for expression of recombinant proteins. BL 41 (DE 3) and BL 43 (DE3) strains
have designed for over express protein where strains contain genetic mutations phenotypically selected for
conferring toxicity tolerance which blocks expression in conventional host system [49]. The strain C41 (DE3)
was derived from BL21 (DE3) [E. coli F- ompT hsdSB (rB- mB-) gal dcm (DE3)]. This strain has been designed
with one uncharacterized mutation that prevents cell death associated with expression of many toxic
recombinant proteins [50]. Another strain C43 (DE3) was derived from C41 (DE3) by selecting for resistance to
a different toxic protein which was not present in C41 (DE3). From the study it was found C43 (DE 3) had
shown ability to express a different set of toxic proteins than C41 (DE3) [51].
          Further, neither strain contains any plasmid or antibiotic resistance markers. However, these two strains
were found different from each other and from BL21 (DE3) by study based on transfection with a plasmid
verification vector, pAVD10. The vector system PAVD10 comprise of uncF gene (it encodes beta-subunit of E.
coli ATPase) under the regulation of T7 promoter. Subsequently, this plasmid was found to lethal for BL21
(DE3) and to induced C41 (DE3), but it is tolerated by C43 (DE3) regardless of induction [52, 53].
          Conventionally E.coli BL21 (DE3) strains and genetically engineered E. coli C41 (DE3) and E.coli
C43 (DE3) strains for over expression contain Lambda DE3 lysogen, which expresses T7 RNA polymerase
from the lacUV5 promoter under IPTG induction. Following strains can be used to express any gene cloned into
a plasmid downstream to T7 promoter [54]. While the engineered strains C41 (DE3) and C43 (DE3) for over
expression posses Chloramphinicol-resistant plasmid that encodes T7 lysozyme, which is a natural inhibitor of
T7 RNA polymerase. These engineered strains which produce T7 lysozyme that suppresses basal expression of
T7 RNA polymerase prior to induction, thus providing additional stability for recombinants encoding
particularly toxic proteins [55 56].

5.2.     Escherichia coli Rosetta Series
         Codon bias in another major problem often encountered while expressing recombinant protein in many
expression host system [57]. During the translation majority of amino acids are encoded by more than one
codons, and each organism carries its own bias in the usage of the 61 available amino acid codons. In every cell,
the population of t-RNA closely reflects the codons bias of the mRNA population [58]. When the mRNA of
heterologous target genes are over expressed in E. coli, differences in codon usage can block translation due to
the demand for one or more t-RNAs that may be rare or lacking in that particular population [59]. Insufficient t-
RNA pools can lead to translational stalling, premature translation termination, translation frame shifting and
amino acid misincorporation [60].
         To overcome with codon bias many attempt have been made by optimizing codons for expression of
recombinant protein. Optimization of codons for each host system is not easy, time consuming and much
complicated. A novel host system has been designed with universal codons which not only allow expression but
also increases expression yield [61]. Rosetta host strains has deigned to fulfill all these requirements and derived
from BL21 derivatives designed to enhance the expression of proteins that contain codons rarely used in E. coli
[62]. The Rosetta strains supply t-RNAs for the codons AUA, AGG, AGA, CUA, CCC, and GGA on a
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compatible chloramphenicol-resistant plasmid, pRARE. Newly Rosetta strains also supply a seventh rare codons
(CGG) in addition to the six found in the original Rosetta strains [63]. By supplying following rare codons,
Rosetta strains provide a ―universal‖ translation, where translation would otherwise be limited by the codons
usage of E. coli. In few Rosetta strains like pLysS and pLacI the rare t-RNA genes are present on the same
plasmids that carry the T7 lysozyme and lac repressor genes respectively [64, 65].




Fig 5: Plasmid pRARE essentially constructed in Rosetta, it enables Rosetta for the expression of protein often
                               get fails in conventional expression host system.

          Simultaneously, pRIG plasmid transformed into pRARE by incorporating rare E. coli codons leuW and
proL t-RNA genes which improve expression level often not present in any of E. coli strains naturally [66].
While plasmid pRARE is capable for encoding t-RNA genes for all of the ―problematic‖ rarely used codons
encoding Arg, Ile, Gly, Leu and Pro, except for Arg CGA/CGG [67]. The entire rare t-RNA assembly was
incorporated to the pLysS and pLacI plasmids which were derived from pACYC184 to construct pLysSRARE
and pLacIRARE, respectively [68]. The plasmids were transformed into various strains to create the Rosetta
series of expression hosts. These host strains enhance protein expression from target genes containing rare E.
coli codons that would otherwise impede translation [69]. Rosetta pLysS and Rosetta-gami-pLysS are two new
variant in this series with slight modifications engineered for over expression of eukaryotic proteins [70].

                  6. Commercially available high level expression host system
         Lack of natural proteins, which used in many areas like therapeutics, industry and analytical triggered
for finding alternative methods for production of these amazing molecules. Commercial demand and industrial
application of recombinant proteins led to develop numbers of new host system and majority of them are in use.
These new generation host system expressed many essential protein in higher amount. Enormous expression
systems are in the commercial market from various producers to achieve desired protein expression. In the
following table the most common host expression systems which are in use summarized as-




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            S. No.          Strain                Company                        Technical information
                                                                        Expression under IPTG Induction Ideal for
               1.        BL21 (DE3)                Novagen                          prokaryotic protein
                                                                                 Contains T7 polymerase
                                                                        Expression under IPTG Induction Ideal for
                         BL21 (DE3)
               2.                                  Novagen                          prokaryotic protein
                           pLysS
                                                                                 Contains T7 polymerase
               3.                                                                Exhibit Over expression
                          C41 (DE3)                 Lucigen               Both prokaryotic protein and eukaryotic
                                                                                          proteins
               4.                                                                Exhibit Over expression
                          C43 (DE3)                 Lucigen               Both prokaryotic protein and eukaryotic
                                                                                          proteins
               5.                                                                 recA- variant of BL 21
                              BLR                  Novagen
                                                                                 Maintain protein stability
               6.                                                       pRARE plasmid offer essential tRNA for
                            Rosetta                 Lucigen
                                                                                    eukaryotic protein
               7.                                                       pRARE plasmid offer essential tRNA for
                        Rosetta pLysS               Lucigen
                                                                                    eukaryotic protein
               8.       Rosetta gami                                    pRARE plasmid offer essential tRNA for
                                                    Lucigen
                          pLysS                                                     eukaryotic protein
               9.         AD494                    Novagen               trx mutant of K 12 Allow protein folding
               10.                                                      Contains pLysS plasmid allow higher level
                         Tuner pLysS               Novagen
                                                                                       of expression

                                                     7. Conclusions
         The recent increasing demand of various protein molecules in industries and therapeutics triggered
development of new technologies to fulfill commercial consumption. Design of high level expression host
systems which not only express recombinant protein to bulk but also provide universal system where different
types of protein can be produced without any complications. Though these engineered expression systems have
made much remarkable contribution in the field of protein expression but still lot of optimization studies needed.
The major challenges which need to achieve these systems are ease of handling, universal application and cost
of these engineered hosts. If we could achieve these challenges, this will make a new era in the molecular
biology regarding commercial production of recombinant proteins.

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